PET-CT and RNA sequencing reveal novel targets for acupuncture-induced lowering of blood pressure in spontaneously hypertensive rats

Manual acupuncture (MA) can be used to manage high blood pressure; however, the underlying molecular mechanism remains unknown. To explore the mechanism of acupuncture in the treatment of hypertension, Wistar Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were subjected to either MA stimulation or the corresponding sham procedure as a negative control (Sham-MA) for 1 week. PET-CT scans, transcriptomics and molecular biology were used to evaluate the effect of MA. The results show that MA can regulate blood pressure in SHRs, change the glucose metabolism of the paraventricular hypothalamus (PVH), and affect the mRNA and protein expression levels of differentially expressed genes in the PVH. These genes may lower blood pressure by regulating angiotensin, endothelial function and inflammation. These findings reveal that MA regulates multiple biological processes and genes/proteins of the PVH, and provide a solid theoretical basis for exploring the mechanisms by which MA regulates hypertension.

www.nature.com/scientificreports/ Quality control for raw data. The raw data for sequencing included low-quality, adaptor-polluted material with a high content of unknown base (N) reads. These reads were removed before data analysis to ensure the reliability of the results. The Q20, Q30, and clean read ratios were calculated, and subsequent analyses were based on these clean reads.
Differential expression analysis. Clean reads were mapped to the reference using Bowtie2 20 , and the gene expression level was calculated with RSEM 21 . DEGs with DESeq2 were detected as requested 22 . DEGs were chosen according to the parameters with a fold change ≥ 2 and an adjusted p-value (p ≤ 0.05). Then, the Pearson correlation between all samples was calculated using cor, and hierarchical clustering between all samples was performed using hclust. Gene expression was compared between SHRs and WKYs, between the MA and SHR groups, and between the Sham-MA and SHR groups. Volcano plots, scatterplots and heatmaps were generated using R version 3.6.0 (https:// cran.r-proje ct. org/ bin/ windo ws/ base/ old/3. 6.0/). The overlap in upregulated and downregulated gene expression between the different groups was analysed using Venn online software VENNY version 2.1 (https:// bioin fogp. cnb. csic. es/ tools/ venny/ index. html). GO and KEGG classification and functional enrichment were performed for all identified DEGs. GO and KEGG analyses were performed using enrichment analysis with the phyper function in R version 3.6.0 (https:// cran.r-proje ct. org/ bin/ windo ws/ base/ old/3. 6.0/).

Real-time PCR.
Eight DEGs were selected for validation using qRT-PCR. cDNA was produced from hypothalamic mRNA (2 μg) using an Invitrogen SuperScript II Reverse Transcriptase reagent kit (Takara, Shanghai, China). OneStep RT-PCR Enzyme mix (Qiagen, Beijing, China) was used for qRT-PCR on an ABI ViiA 7 PCR System (Thermo Fisher Scientific, USA). The Ct value in the reaction was collected using a corrected threshold setting. β-actin was used as the internal reference gene to confirm gene expression levels, and the relative quantification was determined using the 2 −ΔΔCt method. The primers used for qRT-PCR validation are shown in Table 1.
Western blot. Western  Statistical analysis. All data were analysed via SPSS 17.0 (SPSS Inc., Chicago, USA) and expressed as the mean ± SD or median ± interquartile range (IQR) where appropriate. Data with normal distributions were analysed by one-way analysis of variance (ANOVA) or repeated measures analysis of variance, followed by an LSD post hoc test for determining the group differences. Data with nonnormal distributions were analysed using a

Results
Effects of manual acupuncture (MA) on blood pressure (BP). To evaluate the antihypertensive effect of MA, the BP of the rats was measured at 30, 60, and 90 min after the first treatment. As shown in Fig. 1A,B, the SBP and DBP of the MA group were significantly reduced at 30 and 60 min after acupuncture compared with those of the SHR group. Subsequently, for the following 6 days, the BP of the rats was repeatedly measured 30 min after daily acupuncture. Compared with the SHR group, acupuncture at KI3 significantly reduced the SBP and DBP of rats from the first day to the seventh day, as shown in Fig. 1C,D.
Changes in glucose metabolism in the brain of SHRs. Glucose metabolism was significantly increased in the hypothalamus, thalamus, dorsal thalamus and olfactory bulb of the SHR group compared with the WKY group but decreased in other regions including the cingulate cortex, cingulate gyrus, and motor cortex ( Table 2 and Fig. 2A). However, compared with the SHR group, the MA group showed significantly reduced glucose metabolism in the accumbens nucleus, hypothalamus, olfactory bulb, thalamus, anterior commissure, dorsal thalamus, and hypothalamus tuberal regions and increased glucose metabolism in the cerebellum posterior lobe and visual cortex (Table 3 and Fig. 2B). Furthermore, compared with the SHR group, the Sham-MA group showed significantly reduced glucose metabolism in the basal ganglia, caudate putamen, olfactory bulb, prefrontal cortex and orbital cortex, while glucose metabolism was increased in the medulla oblongata (Table 4 and Fig. 2C).   27 Mbp of raw reads were obtained. The percentage of clean reads Q20 and Q30 (the ratio of the quality values of the reads that were respectively larger than 20 and 30, respectively, compared to the total reads) indicated that the sequence was of high quality and could be used for subsequent analyses (  Figure 2. Changes of glucose metabolism in the rat brain. Regional glucose metabolism was scanned after the 7-day treatment. Results are overlaid on an axial view of the rat brain and mapped to the Paxinos and Watson rat brain atlas. (A) SHR group versus WKY group, (B) MA group versus SHR group, (C) Sham-MA group versus SHR group. Color bars represent the t-value of each significant voxel.    Differentially expressed genes in the hypothalamus of WKY, SHR, MA and Sham-MA rats. To study the regulation of DEGs between the WKY, SHR, MA and Sham-MA groups, DEGs in the hypothalamus were analysed. DESeq2 algorithms were based on a negative binomial distribution to detect the DEGs by applying a fold change ≥ 2.00 and an adjusted p-value (p ≤ 0.05). The statistics of the number of DEGs are shown by a heatmap (Fig. 4A,D,G), scatter plot (Fig. 4B,E,H), and volcano plot (Fig. 4C,F,I). There were 695 DEGs in the SHRs relative to the WKY rats, with 375 upregulated genes and 320 downregulated genes. A total of 120 DEGs were found in the MA rats relative to the SHRs, with 72 upregulated genes and 48 downregulated genes. A Venn analysis was used to study the possible genes involved in reducing blood pressure as a result of acupuncture. We found that MA treatment abolished the upregulation of 5 of the 375 genes upregulated in SHRs compared to wild-type rats. Correspondingly, MA treatment was able to counteract the downregulation of 3 of the 320 down- The total amount of reads that contain more than 5% unknown N base (the N reads ratio); adaptor: The total amount of reads that contain adaptors (the adaptor ratio); low quality: More than 20% of the bases in the total read have a quality score lower than 15 (a low quality read ratio); clean reads: Reads filtered with N reads; reads have adaptors and low quality reads (a clean read ratio).   www.nature.com/scientificreports/ regulated genes in SHRs (Fig. 4J,K). However, DEGs that were up-or downregulated by sham acupuncture were not involved in the blood pressure regulation process (Fig. 4L,M).

Gene ontology and Kyoto encyclopedia of genes and genomes analysis.
To investigate the function of the DEGs, Gene Ontology (GO) classification and functional enrichment were performed. GO covers three domains: biological process, cellular component, and molecular function. Functional enrichment was performed and the GO classification results between the WKY, SHR, MA and Sham-MA groups are shown in Fig. 5A-C. Regarding biological processes, the categories "cellular process", "single-organism process", "metabolic process", and "biological regulation" showed a high degree of enrichment. The DEGs were involved in the "cell", "cell part", "organelle", and "membrane" categories according to their cellular component classification. In terms of the molecular functions, "molecular transducer activity", "binding", "catalytic activity", and "nucleic acid-binding transcription factor activity" showed a high degree of enrichment.
To further investigate the possible pathways affected directly by MA treatment in SHRs, DEGs were classified by performing KEGG pathway classification and functional enrichment. The terms with false discovery rates (FDRs) no larger than 0.01 are defined as being significantly enriched. As shown in Fig. 6, DEGs were found to be enriched in several signalling pathways, including "endocrine and metabolic diseases", "neurodegenerative diseases", "cardiovascular diseases", "energy metabolism", and "signaling molecules and interaction".

Validation of the differentially expressed genes using real-time PCR and Western blot.
To verify the reliability of the RNA-Seq data, real-time PCR was used to examine the expression of eight DEGs: Angptl2, Erbb2, Klotho, Ednra, Ccr5, Gnb3, Gpr81, and Cyp1b1. As shown in Fig. 7, trends in the expression data of the examined DEGs were the same as those observed for RNA-Seq. Three genes, Angptl2, Erbb2, and Klotho, were downregulated in the SHR group but upregulated after MA treatment. On the other hand, five genes, Ednra, Ccr5, Gnb3, Gpr81 and Cyp1b1 were upregulated in the SHR group but downregulated after MA treatment. However, Sham-MA did not significantly regulate these DEGs.
To further confirm that the regulated DEGs lead to protein modulation and explain the potential mechanisms underlying the acupuncture effects, western blotting was used to examine the protein levels of the DEGs (Fig. 8). Consistent with the real-time RT-PCR results and as expected, the results of western blotting showed that the protein levels of Ednra, Ccr5, Gnb3, Gpr81 and Cyp1b1 were downregulated in MA-treated rats compared to SHRs, the protein levels of Angptl2, Erbb2 and Klotho were upregulated in the MA group. Interestingly, the protein level of eight DEGs was not regulated after Sham-MA treatment.

Discussion
Essential hypertension is the most common form of hypertension 23 . Although laboratories in many countries have studied the basic mechanism of acupuncture, little is known about its mechanism of regulating blood pressure. Our research results show that within 30 to 60 min after acupuncture treatment, SBP and DBP of SHRs are effectively reduced. Clinical acupuncture usually involves repeated acupuncture treatments. This study also  www.nature.com/scientificreports/ found that the antihypertensive effect of acupuncture can be stably repeated within a 7-day treatment cycle. In fact, repeated acupuncture may produce more molecular changes and longer-term cardiovascular effects than a single bout of acupuncture 24 . Our research shows that acupuncture is a potential method in the treatment of hypertension, and its mechanism is worthy of in-depth study.
To reveal the central mechanism related to the control of blood pressure by acupuncture, we used PET-CT to detect changes in brain glucose metabolism in SHRs. Interestingly, compared with that in the WKY group, the glucose metabolism of PVH in the SHR group was upregulated, and the upregulation was reversed by acupuncture. However, sham acupuncture failed to play a similar regulatory role. Therefore, we speculate that only a few specific acupoints can regulate blood pressure, which is consistent with existing research results 24 . We know that PVH can regulate many human diseases, such as hypertension, obesity and diabetes [25][26][27] . In addition, increased sympathetic outflow from the PVH and excessive activation of sympathetic synapse N-methyl-D-aspartate receptors are critical to hypertension 28 . Our PET-CT results suggest that PVH may be one of the target brain regions for acupuncture KI3 to regulate blood pressure. To further explore the influence of acupuncture on the molecular mechanism affecting the PVH, we then performed PVH transcriptome sequencing.
After statistical analysis of the PVH transcriptome sequencing data, we found that compared with the WKY group, the expression of the Angptl2, Erbb2 and Klotho genes in the PVH of the SHR group decreased, while the expression of Ednra, Ccr5, Gnb3, Gpr81 and Cyp1b1 increased, and acupuncture at KI3 reversed the expression changes of these DEGs. According to the GO functional classification, the DEGs were enriched in the categories of "catalytic activity", "transporter activity", "molecular function regular", and "signal transducer activity". KEGG analysis revealed that the DEGs were involved in several pathways, including the "endocrine system", "cardiovascular diseases", and "neurodegenerative diseases".
However, the exact mechanisms of the involvement of these DEGs in the regulation of blood pressure by acupuncture at KI3 remain unclear. Therefore, we combined the DEGs in the PVH verified by qPCR and western blot in this study with the reported mechanisms of acupuncture-induced lowering of blood pressure to preliminarily clarify the potential central blood pressure regulatory mechanism of acupuncture at KI3.
The DEG Klotho was upregulated in the PVH after MA at KI3. As a newly discovered antiaging gene, Klotho is mainly expressed in the brain and kidney 29,30 . In the kidney, the mechanism by which the Klotho protein regulates blood pressure has been extensively researched. For example, Klotho may exert beneficial antihypertensive activity by inhibiting the activation of the renin-angiotensin system (RAS) and the production of systemic aldosterone. Klotho can inhibit intrarenal RAS by targeting Wnt/β-catenin signalling to prevent Wnt-induced nuclear translocation of β-catenin to lower blood pressure 31 . Researchers found that phosphate may be an independent cause of hypertension, and Klotho is selectively expressed in the distal and proximal tubules to control the reabsorption of phosphate 32 . Recently, researchers have noted the relationship between Klotho and blood pressure regulation in the CNS. The latest research has shown that Klotho can inhibit the level of oxidative stress 33 . Hypertension is related to high levels of oxidative stress in the PVH. Oxidative stress is mainly manifested as an increase in the formation of reactive oxygen species (ROS). This abnormal increase precedes the formation of hypertension in SHRs and is related to the occurrence and maintenance of hypertension 34 . Our previous studies revealed that acupuncture could regulate the expression of 7 proteins related to oxidative stress in the medulla oblongata of SHRs 35 , but the exact regulatory mechanism was still unclear. Based on our results, we speculated that acupuncture at KI3 might upregulate Klotho to inhibit hypothalamic oxidative stress levels, thereby downregulating the blood pressure of SHRs. In addition, Klotho encodes transmembrane glycoproteins with extracellular domains, including Klotho, αKlotho and βKlotho proteins, which are important parts of the endocrine fibroblast growth factor 21 (FGF21) receptor complex 36 . FGF21 has a wide range of biological functions 37 . It can cross the blood-brain barrier and bind to the Klotho protein in the PVH to form a complex. This complex activates the www.nature.com/scientificreports/ HPA axis and regulates the release of corticosterone by acting on hypothalamic neurons. It is worth noting that corticosterone has been widely reported to be related to the suppression of hypertension. According to our data, we speculated that upregulating Klotho in the PVH can promote the formation of the FGF21-Klotho complex, activate the HPA axis to release corticosterone, and ultimately lower blood pressure, which may be a potential central mechanism by which acupuncture at KI3 reduces blood pressure.
Our study showed that acupuncture at KI3 can upregulate the expression of Erbb2 in the PVH of SHRs. The Erbb2 protein is a subtype of the tyrosine kinase receptor family, and it participates in NRG-1/Erbb signalling along with neuromodulin 1 (NRG-1) located in the central nervous system(CNS). Studies have revealed that NRG-1/Erbb signals in the CNS play an important role in cardiovascular homeostasis 38 , and activating the NRG-1/Erbb signalling in the CNS can reduce blood pressure, heart rate and renal sympathetic nerve activity 38 . The expression level of Erbb2 in the brainstem of SHRs was significantly lower than that in WKY rats. Inhibiting the expression of Erbb2 could increase blood pressure and heart rate 39 . Studies have shown that reducing the level of Erbb2 in the rat brain causes hypertension by reducing NO synthesis and inhibiting the activity of γ-aminobutyric acid 40 . According to existing research, Erbb receptors and NRG-1 are widely distributed in the central and peripheral nervous systems 41,42 . Our research showed that, compared with that of WKY rats, the expression of Erbb2 in the PVH of SHRs decreased, while acupuncture at KI3 can upregulated the expression of Erbb2 in the PVH. We speculated that acupuncture at KI3 could upregulate Erbb2 in the PVH, activate NRG-1/ Erbb signalling and inhibit sympathetic nerve activity, thereby decresing blood pressure and heart rate. This may be one of the central mechanisms by which acupuncture regulates blood pressure in SHRs.
CCR5 is a chemokine receptor. Our results showed that CCR5 was highly expressed in the PVH in SHRs, while acupuncture at KI3 could downregulated its expression. Studies have shown that CCR5 can mediate the activation of the NF-κB pathway, while the inflammation mediated by the NF-κB pathway can be inhibited by knocking out or inhibiting the expression of CCR5 43 . Inflammation in the PVH can induce an increase in blood pressure. Under pathological conditions, the PVH drives inflammation through activation of NF-κB, thereby increasing the activity of the renin-angiotensin system, leading to hypertension. In addition, activation of NF-κB can also increase the release of the downstream inflammatory cytokine TNF-α, which in turn increases sympathetic nerve outflow to raise blood pressure. Abnormally elevated TNF-α may also increase the permeability of the blood-brain barrier, allowing more angiotensin to enter the brain, and at the same time the loss of reflex baroreceptor function leading to increased blood pressure 44 . Our results showed that CCR5 in the PVH was inhibited after acupuncture at KI3, which suggested that acupuncture may reduce blood pressure by inhibiting the NF-κB pathway and inflammation of the hypothalamus. On the other hand, CCR5 can also regulate the MAPK pathway. Pharmacological experiments have shown that downregulating CCR5 mediates the degradation of MAPK, IKK and IκBα, and enhances the inhibitory effect of the inflammatory response 45 . Inhibition of MAPK signal transduction in the PVH has been shown to prevent sympathetic nerve overexcitation 46 . Therefore, downregulating the expression of CCR5 in the PVH can improve sympathetic hyperexcitability by inhibiting MAPK-mediated inflammatory pathways, thereby reducing blood pressure. In summary, we believe that the increased expression of CCR5 in the PVH may be one of the mechanisms of the SHR blood pressure increase, and that CCR5 may mediate the activation of the NF-κB and MAPK signalling pathways and the renin-angiotensin system to increase the sympathetic nerve outflow, eventually leading to high blood pressure. In this study, the qPCR and western blot results showed that acupuncture at KI3 downregulated the expression of CCR5, so we speculated that acupuncture may reduce CCR5-mediated inflammation of the PVH and reduce sympathetic nerve outflow to regulate blood pressure.
In addition to the expression of the above genes, that of Angptl2, CYP1B1, GPR81, EDNRA, and GNB3 can also be regulated by acupuncture at KI3. Angiopoietin-like protein 2 (Angptl2) belongs to the angiopoietinlike family. Studies have shown a correlation between the level of circulating Angptl2 and the diagnosis and/or prognosis of cardiovascular disease 47 . Cytochrome P-4501B1 (CYP1B1) induces reactive oxygen species (ROS) to cause inflammation, cardiovascular hypertrophy and hypertension-related endothelial dysfunction, which is mediated by the activation of ERK1/2 and c-SRC signalling pathways 48 . GPR81 belongs to the family of hydroxycarboxylic acid receptors, and existing studies have provided evidence for the potential new role of GPR81 agonists in blood pressure control and renal vascular resistance regulation (including the regulation of the endothelin system, a known vascular effect mechanism). However, the expression level of GPR81 in the brain is low, and it seems to be involved in neuronal signalling 49 . Endothelin receptor type A (EDNRA) combined with vasoconstrictors released by endothelial cells can increase vasoconstriction and sodium retention, leading to increased blood pressure. The use of EDNRA selective antagonists can reduce blood pressure 50 . The G protein β3 (GNB3) subunit is involved in G protein coupled receptor signal transduction. The GNB3 C825T polymorphism is a marker for the treatment of hypertension, and its phenotype has enhanced sodium-proton reverse motility activity. The increase in essential hypertension is related to the T allele of the GNB3 C825T polymorphism 51 . However, to date, no studies have found roles of these genes in central tissues, such as the PVH. Therefore, roles of these DEGs in the PVH and their signalling pathways participating in lowering blood pressure caused by acupuncture at KI3 need to be confirmed in further studies.
One limitation in this study was that the blood pressure measurements were tail-cuff blood pressure measurements instead of radiotelemetry blood pressure measurements. When using this type of measurement, rats may be in a state of stress, which causes the blood pressure measurement value to deviate from the true value slightly.

Conclusion
Our data suggest that acupuncture at KI3 can effectively reduce the blood pressure of spontaneously hypertensive rats. PET-CT scanning results further indicated that the PVH could be one of the target brain areas for MA at KI3 to manage blood pressure. Furthermore, whole transcriptome sequencing of the PVH was used to explore how